Project 1: Muscarinic M5 acetylcholine receptors potentiate synaptic transmission from VTA DA neurons to the NAc (Shin, Adrover, Wess and Alvarez; PNAS 2015) Cholinergic transmission in the striatum functions as a key modulator of dopamine (DA) transmission and synaptic plasticity, both of which are required for reward and motor learning. Acetylcholine (ACh) can elicit striatal DA release through activation of nicotinic ACh receptors (nAChRs) on DA axonal projections. However, it remains controversial how muscarinic ACh receptors (mAChRs) modulate striatal DA release, with studies reporting both potentiation and depression of striatal DA transmission by mAChR agonists. This study investigates the mAChR-mediated regulation of release from three types of midbrain neurons that project to striatum: DA, DA/glutamate, and glutamate neurons. We found that M5 mAChRs potentiate DA and glutamate release only from DA and DA/glutamate projections from the midbrain. We also show that M2/M4 mAChRs depress the nAChR-dependent mechanism of DA release in the striatum. These results suggest that M5 receptors on DA neuron terminals enhance DA release, whereas M2/M4 autoreceptors on cholinergic terminals inhibit ACh release and subsequent nAChR-dependent DA release. Our findings clarify the mechanisms of mAChR-dependent modulation of DA and glutamate transmission in the striatum. Different types of mAChRs (M1-M5) are expressed in the striatum. Gi-coupled M2 receptors are mainly found on presynaptic terminals of CINs and on glutamatergic terminals where they inhibit neurotransmitter release. Gq-coupled M1 receptors are expressed in MSNs, and Gq-coupled M5 receptors are expressed in midbrain DA neurons. Early studies showed that agonists for mAChRs potentiate DA efflux measured in the striatum (de Belleroche and Gardiner, 1982; Lehmann and Langer, 1982; Raiteri et al., 1984). However, DA transients measured using fast-scanning cyclic voltammetry (FSCV) and evoked by electrical stimulation are depressed by mAChR agonists (Kudernatsch and Sutor, 1994; Bendor et al., 2010; Foster et al., 2014). The hypothesis of this study is that agonists for mAChRs have different effects on DA transients evoked by electrical stimulation (eDA) within the NAc compared to DA transients evoked by direct optogenetic activation of DA fibers (oDA) because electrical stimulation drives both ACh-mediated and monosynaptic DA release. This study investigates the modulation of DA transmission by mAChRs when using conventional electrical stimulation and optogenetic stimulation to selectively activate VTA DA neuron fibers. Channelrhodopsin-2 was expressed in DA neurons in the VTA and DA transients were evoked by alternating electrical stimulation with optogenetic stimulation to trigger eDA and oDA transients, respectively. In agreement with previous observations, eDA transients were depressed by 70% by the muscarinic agonist oxotremorine. In contrast, the same concentration of oxotremorine potentiated oDA transients by 20%. These results indicate that a muscarinic agonist has opposite effects on DA transients evoked by electrical stimulation, which involves activation of CINs versus DA transients evoked by optogenetic stimulation that directly activates DA fibers. VTA DA neurons that project to the NAc shell co-release glutamate. Indeed, oxotremorine potentiated oEPSC by 56%) while inhibiting electrically evoked EPSCs recorded on MSNs in the NAc shell, which are largely mediated by activation of cortical synapses that express presynaptic M2 receptors. The potentiation was completely reversed by application of the muscarinic antagonist, scopolamine, and it was totally absent in M5 receptor knockout mice, demonstrating that M5 mAChRs, presumably located in presynaptic DA neurons, are required for the potentiation of neurotransmitter release from DA terminals. Inhibition of acetylcholinesterase activity by application of the selective blocker ambenonium produced robust enhancement of oDA transients, demonstrating that endogenous ACh potentiates DA transmission from midbrain DA terminals. Taken together, our results suggest that the muscarinic receptor M5 potentiates DA and glutamate transmission from midbrain DA neurons in the NAc. Whereas, mAChR agonist decreases eDA transients through activation of mAChRs on CINs, which in turn dampens ACh release and decreases DA and glutamate release from DA terminals. Project: D2 autoreceptors control the reinforcing properties of cocaine and the reactivity to drug-paired cues (Holroyd et al., Neuropsychopharmacology 2015) A prominent aspect of drug addiction is the ability of drug-associated cues to elicit craving and facilitate relapse. In human drug abusers, the presentation of drug-associated cues, such as drug paraphernalia, can trigger large increases in DA in the striatum even in the absence of the drug (Volkow et al., 2006). An abundance of studies have demonstrated a link between low levels of striatal D2 receptors and cocaine abuse, high craving and increased relapse both in humans (Volkow et al., 1997; Volkow et al., 2009) and animal models (Nader et al., 2006; Dalley et al., 2007; Belin et al., 2008). However, the contribution of D2Rs of non-striatal origin to cocaine seeking and the liability for cocaine abuse has been overlooked and is not clearly understood. A recent imaging study in humans showed that low levels of midbrain D2 receptor availability correlates with increased subjective responses to amphetamine and impulsivity traits, two strong predictors for developing substance abuse (Zald et al., 2008; Buckholtz et al., 2010). Animal models and basic research on midbrain D2 receptors in relation to these cocaine behaviors is notably absent. The hypothesis of this study is that D2Rs of non-striatal origin, such as those expressed by midbrain DA neurons, modulate the behavioral responses to cocaine by altering its reinforcing properties and the response to drug-paired cues. D2Rs in DA neurons function as autoreceptors exerting feedback inhibition over DA neuron excitability and DA release. To test this, we took advantage of our recently generated conditional mutant mouse strain selectively lacking D2 autoreceptors (autoDrd2KO) while preserving normal levels of Drd2 mRNA in the striatum (Bello et al., 2011). We showed that selective loss of D2 autoreceptors impairs the feedback inhibition of DA release and amplifies the cocaine effect on DA transients in the NAc. Interestingly, mice lacking D2 autoreceptors were more likely to acquire cocaine self-administration than controls. However, acquisition of cued-operant responding for a food reward was similar in autoDrd2KO mice and controls, indicating a selective vulnerability for psychostimulants. The enhanced acquisition for cocaine seen in autoDrd2KO mice is due to a combination of a higher rate of cocaine infusions and an improved discrimination between the active and inactive holes during the first week of self-administration. The dose dependence of responding for cocaine and the breakpoint values were similar to controls, suggesting that cocaine sensitivity and the motivation to seek cocaine were not altered. Further, autoDrd2KO mice showed a heightened response to cocaine-paired cues, indicating that they attribute enhanced incentive salience to cocaine-paired cues. Cue-induced reinstatement was also stronger in autoDrd2KO mice, suggesting that the enhanced reactivity to cocaine-paired cues is sufficient to trigger drug-seeking behavior even in the absence of the drug, which resembles a critical feature observed in humans that abuse psychostimulants. Altogether, this study shows that D2 autoreceptors can contribute to the vulnerability for cocaine use and relapse by enhancing the reinforcing properties of cocaine and the salience of drug-paired cues.